Renin-inhibitory pepstatin phenyl derivatives

ABSTRACT

A phenyl derivative of pepstatin A, which is much more potent than pepstatin in inhibiting renin enzyme activity and has significantly greater selectivity for renin over pepsin inhibition than does pepstatin, which is useful in treating hypertension and congestive heart failure.

The present invention is concerned with a novel pepstatin phenylderivative of the formula, C₄₀ H₅₉ N₅ O₉, which selectively inhibits theproteolytic enzyme, renin, and is a useful starting material for thepreparation of statine and the benzyl analog of statine; withpharmaceutical compositions containing the novel compound of the presentinvention as an active ingredient; and with methods of treatinghypertension and congestive heart failure and methods of diagnosis whichutilize the novel compound of the present invention.

BACKGROUND OF THE INVENTION

Renin is an endopeptidase secreted by the juxtaglomerular cells of thekidney, which cleaves its plasma substrate, angiotensinogen,specifically at the 10-11 peptide bond, i.e., between Leu 10 and Leu 11in the equinine substrate, as described by Skeggs et al, J. Exper. Med.1957, 106, 439, or between the Leu 10 and Val 11 in the human reninsubstrate, as elucidated by Tewksbury et al., Circulation 59, 60, Supp.II: 132, Oct. 1979. Renin cleaves angiotensinogen to split off thedecapeptide, angiotensin I, which is converted by angiotensin-convertingenzyme to the potent pressor substance angiotensin II. Thus, therenin-angiotensin system plays an important role in normalcardiovascular homeostasis and in some forms of hypertension.

Inhibitors of angiotensin I converting enzyme have proven useful in themodulation of the renin-angiotensin system and consequently, specificinhibitors of the limiting enzymatic step that ultimately regulatesangiotensin II production, the action of renin on its substrate, havealso been sought as effective investigative tools and as therapeuticagents in the treatment of hypertension and congestive heart failure.

Renin antibody, pepstatin, phospholipids, and substrate analogs,including tetrapeptides and octa-toz8 tridecapeptides, with inhibitionconstants (K_(i)) in the 10⁻³ to 10⁻⁶ M region, have been studied.

Many efforts have been made to prepare a specific renin inhibitor basedon pig renin substrate analogy, which as been shown to correlate wellwith and predict human renin inhibitor activity. The octapeptide aminoacid sequence extending from histidine-6 through tyrosine-13 ##STR1##has been shown to have kinetic parameters essentially the same as thoseof the full tetradecapeptide renin substrate.

Kokubu et al., Biochem. Pharmacol., 22, 3217-3223, 1973, synthesized anumber of analogs of the tetrapeptide found between residues 10 to 13,but while inhibition could be shown, inhibitory constants were only ofthe order of 10⁻³ M. Analogs of a larger segment of renin substrate weresynthesized, Burton et al., Biochemistry 14: 3892-3898, 1975, andPoulsen et al., Biochemistry 12: 3877-3882, 1973, but a lack ofsolubility and weak binding (large inhibitory constant) have proven tobe major obstacles to obtaining effective renin inhibitors.

In the case of pepstatin, Umezawa et al., in J. Antibiot. (Tokyo) 23:259-262, 1970, reported the isolated (from culture filtrates ofactinomyces) of that N-acylated pentapeptide, (pepstatin), having thestructure: ##STR2## This pentapeptide was reported to be an inhibitor ofaspartyl proteases such as pepsin, cathepsin D, and renin, with an I₅₀ratio against pepsin and renin generally in the range of 300 to 1000,depending on the sensitivity of the assay. Gross et al., Science175:656, 1972, reported that pepstatin reduces blood pressure in vivoafter the injection of hog renin into nephrectomized rats, but pepstatinhas not found very wide application as an experimental agent because ofits limited solubility and its inhibition of a variety of other acidproteases in addition to renin.

It has now been found that a novel phenyl derivative of pepstatin isabout four times more potent than pepstatin A in inhibiting reninactivity and has an I₅₀ ratio of renin to pepsin which is about ten-foldless than that for pepstatin A. This new phenyl derivative, therefore,offers significant therapeutic advantages for the treatment of highblood pressure and congestive heart failure in mammals. This derivativeis also a useful starting material for the preparation of statine, asynthetic amino acid, which has been successfully substituted into reninsubstrates as a peptide bond isostere at the 10-11 position.

DESCRIPTION OF THE INVENTION

The present invention discloses a new renin-inhibitory pepstatinderivative of the formula: ##STR3## and pharmaceutically-acceptablesalts thereof.

Pharmaceutically-acceptable salts of the Formula I compound include acidaddition salts, such as acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxy-ethanesulfonate, lactate, amleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thicyanate, tosylate, and undecanoate.The base salts of these compounds include ammonium salts, alkali metalsalts such as sodium and potassium salts, alkaline earth metal saltssuch as calcium and magnesium salts, salts with organic bases such asdicyclohexylamine salts, N-methyl-D-glucamine, and salts with aminoacids such as arginine, lysine, and so forth. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride,bromides and iodides; dialkyl sulfates like dimethyl, diethyl anddibutyl; and diamyl sulfates or long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides or aralkylhalides like benzyl and phenethyl bromides and others. Conventionalmethods of preparing these water or oil-soluble or dispersible salts maybe used.

There is further provided in the present invention a pharmaceuticalcomposition for treating renin-associated hypertension and congestiveheart failure, comprising a pharmaceutical carrier, optionally with anadjuvant, and a therapeuticaly-effective amount of the peptide of theformula I. The actual amount of active ingredient that may be combinedwith the carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration, as determined within the professional competence of theattending physician.

The present invention also encompasses the use of the novel peptide offormula I as a starting material for the preparation of the peptidebond-isostere statine and its benzyl analog, by acid hydrolysis andchromatography.

The inhibitor/statine-starting material of the present invention may beprepared by the aerobic fermentation of a new strain of themicroorganism, Streptomyces hygroscopicus (isolated from the forest soilof a small, isolated island of Japan, with a biologically-pure cultureof which being maintained as Merck Culture Collection MA6044, depositedunder the Budapest Treaty with the American Type Culture Collection,Rockville, Maryland, on June 8, 1987, under Accession No. ATCC 53628),or of other natural or artificial mutants or variants derived byphysical or chemical mutagens (such as ultraviolet irradiation ornitrosoguanidine treatment, or recombinat techniques, such as protoplastfusion, plasmid incorporation, gene transfer, and the like) produced orderived from the Streptomyces hygroscopicus culture or of other speciesof the genus, Streptomyces, capable of producing the desired inhibitorcompound.

The cultural characteristics of the producing organism were comparedwith culture descriptions of other Streptomyces species described inBergey's Manual of Determinative Bacteriology, Eighth Edition, 1974,Williams & Wilkens, Baltimore, MD, and the Intenational StreptomycesProject reports: Shirling, E. B.& D. Gottlieb, "Cooperative descriptionof type cultures o f Streptomyces, II. Species description from firststudy", Intern. J. Syst. Bateriol. (IJSB) 18: 69 189, 1968;"III.Additional species descriptions from first and second studies", IJSB 18:279 392, 1968;"IV. Species descriptions from the second, third, andfourth studies", IJSB, 19: 391 512, 1969;"V. Additional descriptions",IJSB 22: 265 394, 1972. By this method, the producing organism wasidentified as a new strain of the known species, Streptomyceshygroscopicus.

The cultural characteristics of this new strain of Streptomyceshygroscopicus (where V=vegetative growth; A=aerial mycelium; andSP=soluble pigment; and all readings were taken after three weeks at 18°C., unless noted otherwise, with the pH of all media approximatelyneutral [6.8-7.2]) include:

Morphology: Sporophores form short compact spirals clustered along theaerial hyphae, and as the culture ages, these spores coalesce to formlarge moist clusters.

Yeast extract-malt extract agar (ISP Medium 2)

V: Reverse - grayish-tan

A: Flat, granular, gray mixed with much white, giving speckledsalt/pepper appearance, edged with dk gray

SP: None

Oatmeal agar (ISP Medium 3)

V: Reverse - dark gray

A: Flat, granular, dark gray mixed with some white

SP: None

Inorganic salts-starch agar (ISP Medium 4)

V: Reverse - dark gray

A: Flat, granular, dark gray, becoming moist as culture ages

SP: None shows hydrolysis of starch

Glycerol asparagine agar (ISP Medium 5)

V: Reverse - dark gray

A: Med. gray mixed with some white, edged in dark gray, flat, granular

SP: None

Peptone-iron-yeast extract agar (ISP Medium 6)

V: Grayish tan

A: None

SP: None

Melanin: Negative

Tyrosine agar (ISP Medium 7)

V: Reverse - grayish tan

A: Light gray mixed with some white, edged with dark gray

SP: None

Czapek-dox agar

V: Reverse - tan

A: Grayish-white, flat, granular

SP: None

Egg albumin agar

V: Reverse - grayish tan

A: Med. gray mixed with white, edged with dark gray, flat, granular

SP: None

Carbon Utilization

Pridham-Gottlieb basal medium (ISP Medium 9)+1% carbon source; +=growth;±=growth poor or questionable; -=no growth as compared to negativecontrol (no carbon source)

Glucose: +

Arabinose: +

Cellulose: -

Fructose: +

Inositol: +

Lactose: ±

Maltose: +

Mannitol: +

Mannose: +

Raffinose: +

Rhamnose: -

Sucrose: +

Xylose: +

Temperature range (Yeast extract-dextrose+salts agar)

28° C. - Good growth and sporulation

37° C. - Poor vegatative growth - no aerial hyphi

42° C. - No growth

50° C. - No growth

Oxygen requirements (Stab culture in yeast extractdextrose +salts agar)

Aerobic

The controlled aerobic fermentation of this new strain of Streptomyceshygroscopius is conducted in a suitable nutrient media which containssources of assimilable carbon (such as from any of a wide variety ofcarbohydrates, including glucose, fructose, maltose, sucrose, xylose,and the like), especially in the presence of nitrogen sources, includingproteinaceous materials (such as yeast hydrolysates, primary yeast,soybean meal, hydrolysates of casein, distillers solubles, cornsteepliquor, tomato paste, amino acids, figs, malts, cottonseed flour, lardwater, animal viscera, and the like) and nutrient inorganic salts (suchas the customary salts capable of yielding sodium, calcium, potassium,cobalt, manganese, iron, magnesium, ammonium, phosphate, sulfate,chloride, carbonate, and the like) at temperatures ranging from about24° to 32° C., optimally at about 28° C., in a pH controlled to 6.8-7.4by the use of suitable organic buffers incorporated into thefermentation medium. Any of a wide variety of media which contain atleast carbon and nitrogen sources (preferably in a ratio of nutritionalnitrogen sources to nutritional carbon sources of from 1:2 to 2:1,particularly in a ratio of from 0.8 to 1.2), with optional additionalnutrients supplied by mineral salts and trace metals, may be used.

The inoculum for the fermentation may come from a small aliquot (seed)of vegetative growth in a seed medium which supports rapid growth of themicroorganism or directly from spores, which is then innoculated into aproduction medium for large scale fermentation under optimum conditions.Usually the maximum yield of the inhibitor is achieved within about 24to 200 hours, particularly in from 24 to 36 hours, although variationsin the medium or in the microorganism will alter the rate of productionand/or its yield. The accumulated products of the fermentation may thenbe separated and recovered from the broth by conventionalchromatographic means.

Such a separation might include filtration of the fermentation broth toseparate mycelia from liquid supernatant. The supernatant is shaken withan equal volume of a moderately polar, water-immiscible solvent, such aschloroform, ethyl acetate, methyl ethyl ketone, and the like, and thelayers are allowed to settle. The mycelia are stirred vigorously(homogenized) with several volumes of solvents, such as acetone, ethylacetate, methyl ethyl ketone, or the like, which will dissolve most ofthe pepstatin phenyl derivatives located within the mycelia. Thecombined mycelia and supernatant organic extracts are concentrated to asmall volume under reduced pressure. The resultant mass is subjected toa series of solvent partioning and washing steps, using petroleum ether,hexane, ether, methylene chloride, methanol and similar solvents.

Adsorption and partition chromatograhies, gel filtration, reversed-phaseliquid chromatography and the like may be used, in conjunction witheluents of proper polarity and solubilizing characteristics to affordthe desired pepstatin phenyl derivative.

The novel peptide of the present invention possesses a high degree ofactivity in treating renin-associated hypertension and congestive heartfailure in humans, as well as in other warm-blooded animals, such asmice, rats, horses, dogs, cats, etc.

Therefore, in accordance with the present invention there is stillfurther provided a method of treating renin-associated hypertension andcongestive heart failure, comprising administering to a patient in needof such treatment, a therapeutically-effective amount of a peptide ofthe formula I.

For these purposes, the peptide of the present invention may beadministered orally, parenterally (including subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques), by inhalation spray, or rectally in dosage unitformulations containing conventional non-toxicpharmaceutically-acceptable carriers, excipients, adjuvants and othervehicles.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleagenous suspension. This suspension may be formulated according tothe known art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

The peptides of this invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable nonirritationgexcipient, such as coca butter or polyethylene glycol, which is solid atordinary temperatures by liquid at the rectal temperature and willtherefore melt in the rectum to release the drug.

Dosage levels of the order of 0.1 to 4.0 grams per day parenterally areuseful in the treatment of the above indicated conditions, with oraldoses three-to-ten times higher. For example, renin-associatedhypertension and hyperaldosteronism are effectively treated parenterallyby the administration of from 1.0 to 50 milligram of the compound perkilogram of body weight per day. It will be understood, however, thatthe specific dose level for any particular patient will depend upon avariety of factors, including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, rate of excretion, drugcombination and the severity of the particular condition undergoingtherapy.

The renin-inhibitory peptides of the present invention may also beutilized as diagnostic methods for the purpose of establishing thesignificance of renin as a causative of contributory factor inhypertension or congestive heart failure in a particular patient.

Both in vivo and in vitro methods may be employed. In the in vivomethod, a novel peptide of the present invention is administered to apatient. in a single dose of from 0.1 to 10 mg per kg of body weight,preferably by intravenous injection, although other routes of parenteraladministration are also suitable, at a hypotensive dosage level and as asingle dose, and there may result a transitory fall in blood pressure.This fall in blood pressure, if it occurs, then indicates supranormalplasma renin levels.

The following Examples are intended to be representative and notlimiting.

EXAMPLE 1

A frozen spore stock of the culture ATCC (Merck Culture CollectionMA6044) was prepared by suspending the contents of a lyophilizedpreparation in 1 ml of sterile distilled water and applying its contentto the surface of several BYME agar plates.

    ______________________________________                                        BYME Agar                                                                     ______________________________________                                        Yeast Extract (Difco)    4.0 g/L                                              Malt Extract (Difco)     10.0 g/L                                             Glucose                  4.0 g/L                                              3-N-(Morpholino)-propane-sulfonic                                                                      5.8 g/L                                              acid                                                                          pH 7.2                                                                        ______________________________________                                    

These plates were incubated at 28° C. for 10-14 days, with the resultingspores being harvested and suspended in buffered 0.5% methylcellulose,prior to freezing at -80° C.

A frozen vial containing the spores o f the culture MA6044 was thawedand 0.1 ml of its contents used ot inoculate a 250 ml baffled flaskcontaining 50 ml of KE medium.

    ______________________________________                                        KE                                                                            ______________________________________                                        Glucose         1.0 g/L                                                       Dextrin         10.0 g/L                                                      Beef Extract    3.0 g/L                                                       Ardamine pH     5.0 g/L                                                       NZ Amine ε                                                                            5.0 g/L                                                       MgSO.sub.4 ·7H.sub.2 0                                                               0.05 g/L                                                      Phosphate Buffer                                                                              2.0 ml                                                        CaCO.sub.3      0.5 g/L                                                       pH 7.0-7.2                                                                    ______________________________________                                        Phosphate Buffer                                                              ______________________________________                                        K.sub.2 HPO.sub.4                                                                             91.0 g/L                                                      Na.sub.2 HPO.sub.4                                                                            95.0 g/L                                                      pH 7.0                                                                        ______________________________________                                    

This seed was incubated at 28° C. for 2 days at 220 rpm.

A 2% seed inoculum was then used to inoculate a 3 L stirred fermentationvessel containing R2 medium

    ______________________________________                                        R2                                                                            ______________________________________                                        Glucose                  19.0 g/L                                             Yeast Extract (Difco)    7.0 g/L                                              Edamine (Sheffield)      7.0 g/L                                              Amisoy (Sheffield)       7.0 g/L                                              Malt extract (Difco)     6.0 g/L                                              NaH.sub.2 PO.sub.4       1.4 g/L                                              N--2-hydroxyethylpiperazine-N'--2-                                                                     13.0 g/L                                             ethanesulfonic acid                                                           pH 7.0                                                                        ______________________________________                                    

in which sterile glucose had been added after the medium was sterilizedby steam treatment. The vessel was kept at 28° C. and stirred at 500rpm, with 0.75 L/min air flow for 48 hours, with the remainder of thefermentation period being at 700 rpm, with 1.25 L/min air flow.

The inhibitor prepared according to this procedure demonstrated 97%renin inhibition, at a 1:10 dilution with 50% methanol, within 32 hours;and the titer remained at 91-100% inhibition throughout the remainingincubation period.

EXAMPLE 2

A frozen vial containing the spores of Merck Culture Collection MA6044was thawed and 0.1 ml of its contents used ot incoculate a 250 mlbaffled flask containing 50 ml of R2 medium. This medium was incubatedat 28° C. for 4 days at 220 rpm and at the end of the incubation periodthe broths were placed into a equal volume of methanol and centrifugedprior to assay. All samples were diluted with 50% methanol to determinetiter endpoint.

Samples produced according to this procedure exhibited 90% renininhibition at a 1:4 dilution.

EXAMPLE 3

Forty liters of whole broth were acidified with concentratedhydrochloric acid to pH 2.8 and extracted twice with 12 liters of ethylacetate. The combined soluble extracts were concentrated in vacuo todryness, and the residue was washed with hexane and redissolved inmethanol.

The methanol solution was again concentrated to dryness and the residueagain redissolved in methanol at 102.4 mg/ml then diluted with water to12.5 ml (pH 2.8) and extracted sequentially with 100 ml of hexane twice,with 100 ml of methylene chloride twice and 100 ml of ethyl acetatethree times. The combined ethyl acetate extracts were dried over sodiumsulfate, filtered and dried in a vacuum.

The residue was redissolved in methanol to about 50 ml, with 35 mgresidue/ml, and the solution was passed through an LH-20 column and theactive fraction eluted with 10:1 ethyle acetate/MeOH. This fraction wasthen pooled and further purified by Zorbax reverse phase C₁₈ highperformance liquid chromatography, with the active fraction from theZorbx column being eluted with 42.5% acetonitrile/0.2% acetic acid,collected and dried.

The yield of the purified component was 4.6 mg.

Employing low resolution mass spectra and high resolution massmeasurements recorded on a Finnigan-MAT212 mass spectrometer in theelectron impact mode (EI, 90 eV) and positive Fast Atom Bombardment[(+)FAB] spectra obtained on a MAT731 mass spectrometer, as well as a ¹H-NMR spectrum recorded at room temperature on a Varian XL-400 NMRspectrometer in CD₃ OD, and comparing the resulting spectral data tothose of known pepstatins, a compound of Formula I was identified asthat purified component.

EXAMPLE 4

The compound of Example 1 was evaluated against renin, employing a solidphase radioimmunoassay adapted and modified from the assay of Ikedo etal., W. of Clinical Endocrinology and Metabolism, 54, 423-428, 1982),and pepsin assays as illustrated below:

A. Renin Assay Methodology Ten μl of the test inhibitor/broth extractfrom Example 1 was mixed with 5 μl of 0.11M phenylmethylsulfonylfluoride and 0.25 ml of 0.1M potassium phosphate buffer, pH 7.0,containing 0.05% sodium azide and one mg/ml of bovine serum albumin.Twenty μl of 2 μg renin enzyme prepared from hog kidney was added andthe mixture was incubated at 37° C. for 20 minutes. The enzyme reactionwas initiated by adding 20 μl of substrate solution which was preparedby dissolving 4 mg of angiotensinogen in 4 ml of 0.1M potassiumphosphate buffer, pH 7.0, containing 0.05% sodium azide and 1 mg/ml ofBSA.

After 20 minutes incubation at 37° C., the enzyme reaction was stoppedby the addition of 100μl of ¹²⁵ I-angiotension I tracer-pepstatin. Asolution which was prepared by mixing 10 μl of 0.5 mg ¹²⁵ I-angiotensinI containing 200 μl of 16 mg/ml of pepstatin A in dimethyl formamide andeight ml of 0.1M potassium phosphate buffer, pH 7.0, and 0.05% sodiumazide. One antibody coated ball was then added to each assay tube andthe tube was vortexed and incubated at room temperature for 3 hours. Twoml of water was then added ot each tube and the solution was aspirated,with the tube with the ball being counted in a γ-counter.

The activity of the test compound was calculated based on the standardcurve of known amount of angiotensin I.

BN Pepsin Assay Methodology Seventy-five μl of 50% aqueous MeOH extractof the test broths/inhibitor from Example 1, by 0.4 ml of 0.06N HCl and25 μl of 0.15 μg pepsin was added ot a test tube, mixed and incubated at37° C. for 10 minutes. The enzyme reaction was initiated by adding 0.5ml of ¹⁴ C-hemoglobin substrate containing 0.2 mg of hemoglobin and0.003 μg of ¹⁴ C-methemoglobin.

After 45 minutes incubation at 37° C., 0.1 ml of 2% hemoglobin in 0.6NHCl was added to the above solution and the enzyme reaction was stoppedby adding 0.5 ml of 10% trichloroacetic acid. The tube was mixed andcentrifuged for 5 minutes at 500 xg. One ml of supernatant was mixedwith 10 ml of scintillation fluid and the radioactivity was counted.

The activity for the test compound was calculated by the percentage of¹⁴ C-hemoglobin being solubilized.

C. Test Results and Analysis The compound of Example 1 shows adose-related inhibition against both enzymes. The I₅₀ for the compoundof Example 1 in renin was estimated to be 70 ng/ml which is about fourtimes more potent than that of pepstatin A. The I₅₀ for the compound ofExample 1 in pepsin was estimated to be 0.56 ng/ml which is aboutone-half of the activity of pepstatin A.

Thus, the I₅₀ ratios of both compounds against renin and pepsin assaysare summarized below:

    ______________________________________                                                      I.sub.50 ratio of renin/pepsin                                  ______________________________________                                        Compound of Example 1                                                                          125                                                          Pepstatin A     1160                                                          ______________________________________                                    

The I₅₀ ration of the compound of Example 1 is about ten times betterthan that of pepstatin A.

What is claimed is:
 1. A compound of the formula ##STR4## or apharmaceutically-acceptable salt thereof.
 2. A composition comprising atherapeutically-effective amount for treating renin-associatedhypertension or congestive heart failure of a compound according toclaim 1, and a pharmaceutical carrier.
 3. A composition according toclaim 2 also comprising an adjuvant.
 4. A method of treatingrenin-associated hypertension or renin-associated congestive heartfailure comprising administering to a patient in need of such treatment,a therapeutically-effective amount of the compound according to claim 1.5. A method according to claim 4, wherein the therapeutically-effectiveamount is from 0.1 to 4.0 gm/day.
 6. A method of identifying renin as acausative factor in patients suffering from hypertension comprisingadministering to such a patient between 0.1 and 10 mg of body weight ofa compound according to claim 1 and measuring the fall in blood pressureof said patient.